Compositions for Use in Treatment of Chlamydia

This application is a continuation of U.S. patent application Ser. No. 18/594,291, filed Mar. 4, 2024, now U.S. Pat. No. 12,263,213, which claims the benefit of U.S. Provisional Patent Application Serial Nos. 63/449,571, filed Mar. 2, 2023, and 63/500,393, filed May 5, 2023, European Patent Application Nos. 23305375.0, filed Mar. 20, 2023, and 23306372.6, filed Aug. 11, 2023, the entire disclosures of which are hereby incorporated herein by reference.

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file, created on Jul. 25, 2025, is named 761900_SA9-373CON_ST26.xml and is 1,588,301 bytes in size.

The invention is in the field of treating and preventinginfections. In particular, the invention relates to antigens and antigen combinations which can be used as vaccines to immunise againstsp. infection. The vaccines may be delivered as nucleic acids (e.g., mRNAs) encoding antigenic proteins or as recombinant protein antigens.

Chlamydiae are intracellular bacterial pathogens responsible for a variety of infections, including the human sexually transmitted disease and eye infections (Trachoma) caused by. The genusfurther comprises the species, and

() comprises three biovars which lead to an array of pathological conditions in humans, and which are further subdivided into serovars.Serovars A-C (A, B, Ba, and C), of the trachoma biovar, can causeconjunctivitis or trachoma, a disease that can result in blindness.Serovars D-K (D, E, F, G, H, I, J, and K), of the genital tract biovar, cause disease in the genital tract.The lymphogranuloma venereum biovar, serovars L1-3 (L1, L2, and L3), causes invasive urogenital and anorectal infection and has become particularly associated with HIV-infected men who have sex with men.

(serovars D-K) is the most common bacterial agent of sexually transmitted infections.In 2020, the WHO estimated 129 million newinfections. These are of public health concern particularly because untreated infections are often asymptomatic or with minimal symptoms, but they contribute to the transmission of the pathogen. Furthermore, if left untreated, the infection can, amongst others, lead to salpingitis, endometritis, pelvic inflammatory disease (PID), ectopic pregnancy, tubal factor infertility, and can increase the risk for the transmission or acquisition of HIV and the development of cervical carcinoma.Whileinfections can be effectively controlled by antibiotic therapy, due to the high prevalence of cases and their frequently delayed diagnosis for asymptomatic cases,has become a leading cause of female infertility worldwide.There is a need for developing an effectivevaccine as a sustainable strategy to curb infection rates of

The present inventors have providedsp. antigens and antigen combinations which can be used to immunise againstsp.

In particular, the inventors found that antigens derived from a native major outer membrane protein (MOMP) polypeptide ofandantigens other than MOMP elicited robust T cell responses, particularly induction of IFN-γ-producing CD4+ T cells, and/or B cell (i.e. antibody) responses when delivered by mRNAs encoding the relevant antigens. In particular, T cell responses are considered critical for protective immunity.

Accordingly, the invention provides modified MOMP polypeptides, chimeric MOMP VD polypeptides and CT443, CT584, CT600 and CT812sp. polypeptides and nucleic acids comprising a nucleotide sequence encoding such polypeptides.

Polypeptide antigens described herein may be delivered by, i.e. in the form of, a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding said polypeptide.

MOMP is an integral membrane porin proteinfound in bacteria ofsp. It makes up roughly 60% of the protein mass in themembraneand contains multiple B & T cell epitopes.Ininfection, MOMP is an immunodominant antigen. Native MOMP polypeptides ofsp. include five conserved domains separated by four surface exposed variable domains (VD1, VD2, VD3 and VD4).Thus, in a native MOMP polypeptide ofsp., VD1, VD2, VD3 and VD4 are surface exposed loops. Withinspecies (e.g.,), native MOMP polypeptides vary in their VD sequences across different serovars.Within each serovar of, native MOMP polypeptide sequences are highly conserved with greater than 98% sequence identity. Across serovars of, the protein sequences are highly conserved, with exception of the surface exposed VDs. Neutralising antibody epitopes have been mapped to VDs.

The inventors have recognised that IFN-γ producing CD4+ T cells are critical for protective immunity; indeed cellular IFN-γ production has been shown to strongly correlate with protectionand CD4+ T cells appear to be critical since HIV+ women are at increased risk forreinfection. There is also evidence thatantibodies (i.e. B cell-mediated responses) alone may not prevent reinfection and the presence of anti-IgG may even enhance the risk for incident infection.Anti-antibodies have been shown to accelerate clearance of a primarychallenge in the presence of CD4+ T cells in mice, however, in the absence of CD4+ T cells, the primary challenge was not cleared.

A “nativesp. MOMP polypeptide” as used herein encompasses a mature form of a full-length native MOMP polypeptide of asp. without its native signal peptide sequence and a full-length native MOMP polypeptide of asp. including its native signal peptide sequence. In exemplary embodiments, thesp. referred to herein is. A nativeMOMP polypeptide may be of one of serovars A-C, D-K or L1-L3, e.g., serovars D-K. In some embodiments, the nativeMOMP polypeptide is of one of serovars D-G. In some exemplary embodiments, the nativeMOMP polypeptide is of serovar E. Exemplary sequences of nativesp. MOMP polypeptides comprising the native signal peptide sequence are provided by SEQ ID NOs 1-4.

The amino acid sequence of a nativeMOMP polypeptide of serovar E is provided in SEQ ID NO: 2:

The native signal peptide sequence corresponds to residues 1-22 in the amino acid sequence of MOMP ofserovar E (SEQ ID NO: 2) and is shown underlined. Residues 23-393 of SEQ ID NO: 2 correspond to the mature form of full-length native MOMP polypeptide of serovar E ofwhich lacks the native signal peptide sequence. VD1-VD4 sequences of the MOMP polypeptide of serovar E ofare shown in bold and underlined text above.

A nativesp. MOMP polypeptide may comprise the sequence of:

A nativesp. MOMP polypeptide comprises five conserved domains separated by four variable surface exposed domains (VD1, VD2, VD3 and VD4). For example, in the native MOMP polypeptide ofserovar E, VD1 (SEQ ID NO: 9) corresponds to amino acid residues 86-105 of SEQ ID NO: 2, VD2 (SEQ ID NO: 10) corresponds to amino acid residues 161-181 of SEQ ID NO: 2, VD3 (SEQ ID NO: 11) corresponds to amino acid residues 245-260 of SEQ ID NO: 2, and VD4 (SEQ ID NO: 12) corresponds to amino acid residues 309-337 of SEQ ID NO: 2. Conserved domains correspond to amino acid residues 23-85 (first conserved domain); 106-160 (second conserved domain); 182-244 (third conserved domain), 261-308 (fourth conserved domain) and 338-393 (fifth conserved domain). For serovar E, either VD4 (SEQ ID NO: 12) corresponds to positions 309-337 in SEQ ID NO: 2 and the fifth conserved domain corresponds to positions 338-393 in SEQ ID NO: 2 or VD4 (SEQ ID NO: 883) corresponds to positions 309-338 in SEQ ID NO: 2 and the fifth conserved domain corresponds to positions 339-393 in SEQ ID NO: 2.

For serovars D, F and G of, the residues corresponding to VD1, VD2, VD3 and VD4 and the five conserved domains are set out in Table 1. For serovar D, either VD4 (SEQ ID NO: 8) corresponds to positions 309-337 in SEQ ID NO: 1 and the fifth conserved domain corresponds to positions 338-393 in SEQ ID NO: 1 or VD4 (SEQ ID NO: 882) corresponds to positions 309-338 in SEQ ID NO: 1 and the fifth conserved domain corresponds to positions 339-393 in SEQ ID NO: 1. For serovar F, either VD4 (SEQ ID NO: 16) corresponds to positions 310-339 in SEQ ID NO: 3 and the fifth conserved domain corresponds to positions 340-395 in SEQ ID NO: 3 or VD4 (SEQ ID NO: 884) corresponds to positions 310-340 in SEQ ID NO: 3 and the fifth conserved domain corresponds to positions 341-395 in SEQ ID NO: 3. For serovar G, either VD4 (SEQ ID NO: 20) corresponds to positions 310-339 in SEQ ID NO: 4 and the fifth conserved domain corresponds to positions 340-395 in SEQ ID NO: 4 or VD4 (SEQ ID NO: 885) corresponds to positions 310-340 in SEQ ID NO: 4 and the fifth conserved domain corresponds to positions 341-395 in SEQ ID NO: 4.

NativeMOMP polypeptides of serovars A-C, H-K, or L1-L3, or other nativesp. MOMP polypeptides comprise VD1-VD4 and conserved domains at positions corresponding to the residue numbering set out for the native MOMP polypeptides of serovars D-G of, e.g., serovar E.

The sequences of other nativesp. MOMP polypeptides, including those ofMOMP polypeptides of serovars A-C, H-K, or L1-L3 are well known and available from public databases. For example, amino acid sequences of nativesp. MOMP polypeptides are available on the Uniprot (uniprot.org/) and NCBI (ncbi.nlm.nih.gov/) databases. Nucleic acid sequences encoding nativesp. MOMP polypeptides may be obtained from the NCBI (ncbi.nlm.nih.gov/) database. The positions of VDs and conserved domains within other nativesp. MOMP polypeptides are well known and in any event can be determined by aligning sequences of e.g.,serovars D, E, F, and G with sequences of other nativesp. MOMP polypeptides and looking for regions of high sequence conservation to identify e.g., conserved domains.

The inventors have generated modified MOMP polypeptides comprising non-native loop sequences instead of nativesp. MOMP VD sequences. The inventors have recognised that such modified MOMP polypeptides can be used to elicit a protective immune response againstsp. infection. In particular, the inventors have demonstrated that such modified MOMP polypeptides induce a T cell response, including CD4+ T cells such as IFNγ-producing CD4+ T cells. Notably, IFN-γ producing CD4+ T cells are thought to be important in protecting againstsp. infections. Elimination of native VD sequences may therefore reduce B cell-mediated (e.g., antibody) responses (e.g., to B cell epitopes in VDs), while prioritising T cell responses to MOMP, e.g., to sequences in the conserved domains which are conserved between serovars. The inventors have shown that modified MOMP polypeptides can elicit a cross-serovar T cell immune response, i.e., a T cell immune response that is cross-reactive against two or more serovars ofsp., e.g., serovars D-G and J of

The inventors have therefore demonstrated that these modified MOMP polypeptides are suitable vaccine candidates that may be used either as stand-alone antigens or in combination with othersp. antigens that can elicit B cell-mediated (e.g., antibody) responses and/or T cell-mediated responses in a subject, such as one or more chimeric MOMP VD polypeptides provided herein and/or one or moresp. CT443, CT584, CT600, or CT812 polypeptides provided herein, in order to promote long-lasting memory T cell responses and protective immunity against infection. A modified MOMP polypeptide as described herein may be delivered by a nucleic acid comprising a nucleotide sequence encoding the modified MOMP polypeptide.

Accordingly, in one aspect, the invention provides a nucleic acid comprising a nucleotide sequence encoding a modified MOMP polypeptide, wherein the modified MOMP polypeptide has an amino acid sequence comprising two or more conserved domain sequences of a nativesp. MOMP polypeptide and a non-native loop sequence between the conserved domain sequences. In a further aspect, the invention provides a modified MOMP polypeptide having an amino acid sequence comprising two or more conserved domain sequences of a nativesp. MOMP polypeptide and a non-native loop sequence between the conserved domain sequences.

In an exemplary embodiment, the modified MOMP polypeptide does not comprise a nativesp. MOMP variable domain between the two or more conserved domain sequences. Thus, in a modified MOMP polypeptide, a non-native loop sequence may replace a nativesp. MOMP polypeptide VD sequence. A modified MOMP polypeptide may therefore omit a nativesp. MOMP VD sequence between the two or more conserved domain sequences and include a non-native loop sequence between the two or more conserved domain sequences.

Replacing nativesp. MOMP VD sequences with non-native loops removes B cell epitopes (e.g., antibody epitopes) found in the nativesp. MOMP VDs. Removal of these B cell epitopes may reduce B cell-mediated responses to the antigen in a subject (e.g., to B cell epitopes in native MOMP VDs). This may advantageously increase T cell responses to MOMP in a subject, e.g., to sequences in the MOMP conserved domains which share a high degree of identity between MOMP serovars. The modified MOMP polypeptides of the invention may therefore elicit a cross-serovar T cell immune response in a subject, i.e. a T cell immune response that is cross-reactive against two or more serovars ofsp. (e.g., cross-reactive against two or more serovars (e.g., serovars D-G and J) of). Thus, the modified MOMP polypeptides of the invention may be capable of enhancing T cell-mediated responses (e.g., relative to a corresponding native MOMP polypeptide). The modified MOMP polypeptides of the invention may be capable of enhancing a CD4+ T cell-mediated response (e.g., a IFNγ+CD4+ T cell response such as IFNγ+IL2+ TNFα+CD4+ T cell response) e.g., relative to a corresponding native MOMP polypeptide. The modified MOMP polypeptides of the invention may be capable of inducing a T cell response (e.g., an antigen specific T cell response) in a subject. The T cell response may be immunodominant. Thus, the modified MOMP polypeptide of the invention may be capable of eliciting a T cell population responsive to a nativesp. MOMP polypeptide in a subject. In some embodiments, the T cell response is a CD4+ T cell response (e.g., IFNγ+CD4+ T cell response). In some embodiments, the T cell response is a IFNγ+IL2+ TNFα+CD4+ T cell response.

“Non-native loop sequences” refer to sequences that are not native to anysp. MOMP VD (VD1, VD2, VD3 or VD4) of any serovar. In some embodiments, non-native loop sequences are no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10% or no more than 5% identical to any nativesp. MOMP VD (VD1, VD2, VD3 or VD4) sequences of any serovar. In some embodiments, non-native loop sequences are no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10% or no more than 5% identical to MOMP VD sequences (VD1, VD2, VD3 or VD4) of serovar E of(SEQ ID Nos: 9-12).

Non-native loop sequences may allow the conserved domain sequences in the modified MOMP polypeptide to form a beta-barrel structure, e.g., to maintain a beta-barrel structure of the conserved domains of a nativesp. MOMP polypeptide. Thus, in the modified MOMP polypeptides of the invention, the two or more conserved domain sequences are linked by a non-native loop sequence such that the conserved domain sequences form a beta-barrel structure (e.g., made up of anti-parallel beta strands). The beta-barrel structure may be as predicted in silico (e.g., using Alphafold2 (Deepmind) software).

In silico modelling may be used to create a 3D structure of a native MOMP protein, which comprises a beta barrel with VD1-VD4 sequences as loops linking individual beta strands. The VD1-VD4 loops are located on the same side of the barrel structure (surface exposed in a native MOMP polypeptide). Non-native loop sequences may be mapped onto VD loops in an in silico model and suitable non-native loop sequences may be determined by the skilled person. Suitable non-native loop sequences include peptide sequences that can cover the distance between the ends of the conserved domains flanking a native VD, e.g., as modelled in silico. The distance may correspond to the distance in e.g., angstrom as determined by in silico 3D model between the last conserved domain residue before the VD sequence to the first conserved domain residue after the VD sequence.

Non-native loop sequences may exclude sequence motifs that are found in a subject (e.g., human) proteome, e.g., sequence motifs of 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more or 15 or more amino acids in length. Excluded subject proteome (e.g., human proteome) sequence motifs are typically 8 or more amino acids in length. This helps minimise unwanted cross-reactivity due to homology between the antigen and self protein.

A non-native loop sequence may be between 3 and 30 amino acids in length, e.g., between 4 and 20 amino acids in length. In some embodiments, a non-native loop sequence replacing VD1 may have a sequence according to SEQ ID NO: 462 or 466 (e.g., SEQ ID NO: 462). In some embodiments, a non-native loop sequence replacing VD2 may have a sequence according to SEQ ID NO: 463 or 467 (e.g., SEQ ID NO: 463). In some embodiments, a non-native loop sequence replacing VD3 may have a sequence according to SEQ ID NOs 464 or 468 (e.g., SEQ ID NO: 464). In some embodiments, a non-native loop sequence replacing VD4 may have a sequence according to SEQ ID NO: 465 or 469 (e.g., SEQ ID NO: 465). Typically, a modified MOMP polypeptide may comprise four non-native loop sequences according to SEQ ID NOs 462-465 in place of VD1, VD2, VD3 and VD4, respectively. A modified MOMP polypeptide may alternatively comprise four non-native loop sequences according to SEQ ID NOs 466-469 in place of VD1, VD2, VD3 and VD4, respectively.

A “conserved domain sequence of a nativesp. MOMP polypeptide” refers to a sequence of a conserved domain of a MOMP polypeptide of aspecies of any serovar, or a variant thereof. Variants include sequences having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity (e.g., at least 95% identity) to a conserved domain of a MOMP polypeptide of asp. of any serovar. In some embodiment, the conserved domain sequences of the modified MOMP polypeptide is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical (at least 95% identical) a conserved domain of a native MOMP polypeptide of asp. of any serovar (e.g., a conserved domain as defined in Table 1). Variants also include truncated forms of native conserved domains of a MOMP polypeptide of asp., for example wherein a conserved domain sequence in a modified MOMP polypeptide lacks up to 3, 5, 8, 10, 15, 20, 25 or 30 (e.g., up to 3 or 5, e.g., up to 3) amino acids of a nativesp. MOMP conserved domain sequence. In some embodiments, the conserved domain sequences of the modified MOMP polypeptide lack up to 3 or 5 amino acids of a nativesp. MOMP conserved domain sequence. The conserved domain boundaries forserovars D, E, F and G are set out in Table 1.

In some embodiments, the modified MOMP polypeptide comprises two, three, four or five conserved domains of a nativesp. MOMP polypeptide, or variants thereof. In some embodiments, a modified MOMP polypeptide comprises five conserved domain sequences of a nativesp. MOMP polypeptide, or variants thereof. In some embodiments, the modified MOMP polypeptide comprises all five full-length conserved domains of a nativesp. MOMP polypeptide. The conserved domains of the modified MOMP polypeptide may collectively have at least 95% sequence identity to the conserved domains of a native MOMP polypeptide (e.g., serovar E MOMP). In some embodiments, the conserved domain sequences of the modified MOMP polypeptide collectively have at least 90% or at least 95% sequence (e.g., at least 95%) identity to the conserved domains of a native MOMP polypeptide (e.g., the conserved domains as defined in Table 1).

Conserved domains of a modified MOMP polypeptide may comprise conserved domain sequences of MOMP from a serovar of(e.g., serovars D-K, such as serovars D-G). In some embodiments, conserved domains of a modified MOMP polypeptide may comprise conserved domain sequences of MOMP from serovar E of

A modified MOMP polypeptide may comprise a non-native loop sequence between each of the conserved domain sequences. Thus, a modified MOMP polypeptide may comprise one, two, three or four non-native loop sequences. The one or more non-native loop sequence(s) may replace one, two, three or four of the corresponding native VD loops. Native VDs may be retained between any remaining conserved domains. The modified MOMP polypeptide may have five conserved domain sequences of a nativesp. MOMP polypeptide. In some embodiments, the modified MOMP polypeptide does not comprise any nativesp. MOMP VDs between any of the conserved domain sequences. In some embodiments, the modified MOMP polypeptide comprises four non-native loop sequences and does not comprise any nativesp. MOMP VDs between the conserved domain sequences. In some embodiments, the modified MOMP polypeptide does not comprise any nativesp. MOMP VDs (or fragments of at least 5, 6, or 7 consecutive amino acids thereof).

In some embodiments, the modified MOMP polypeptide comprises (1) all five conserved domain sequences of asp. MOMP polypeptide ofof any serovar, and (2) four non-native loop sequences, wherein a non-native loop sequence is located between each of the conserved domain sequences, and wherein the modified MOMP polypeptide does not comprise any nativesp. MOMP variable domains between any of the conserved domain sequences, further wherein the non-native loop sequences are between 3 and 30 amino acids in length and no more than 40% identical to any nativesp. MOMP VD (VD1, VD2, VD3 or VD4) sequences of any serovar.

A modified MOMP polypeptide may comprise an amino acid sequence (e.g., from N terminus to C terminus) according to formula:

wherein C1 and C2 are two conserved domain sequences of a nativesp. MOMP polypeptide and L1 is a non-native loop sequence.

In some embodiments, the modified MOMP polypeptide comprises an amino acid sequence (e.g., from N terminus to C terminus) according to formula:

wherein C1, C2 and C3 are three conserved domain sequences of a nativesp. MOMP polypeptide and L1 and L2 are non-native loop sequences.

In some embodiments, a modified MOMP polypeptide comprises an amino acid sequence (e.g., from N terminus to C terminus) according to formula:

wherein C1, C2, C3 and C4 are four conserved domain sequences of a nativesp. MOMP polypeptide and L1, L2 and L3 are non-native loop sequences.

In some embodiments, a modified MOMP polypeptide comprises an amino acid sequence (e.g., from N terminus to C terminus) according to formula:

wherein C1, C2, C3, C4 and C5 are five conserved domain sequences of a nativesp. MOMP polypeptide and L1, L2, L3 and L4 are non-native loop sequences.

The conserved domain sequences and non-native loop sequences may be as described herein.